RNA Sample Preparation
RNA Sample Submission Guidelines
The success of an RNA project is primarily dependent on the quality of the material received
from the collaborator. JGI currently uses the Illumina platform (RNA-seq) for all RNA-based
projects, including genome annotation and gene expression profiling projects. If you have any
questions or concerns, contact your Project Manager or the Project Management Office.
Samples not meeting minimum submission requirements with regard to quantity and
quality will result in a delay to project initiation.
View protocols for isolating RNA, provided by the JGI user community.
RNA sample requirements:
The JGI uses total RNA as starting material for all of its library protocols.
Different types of samples may require specific RNA isolation techniques. JGI has successfully
used standard methods involving Trizol, Rneasy and for many samples further purified using
LiCl. Contaminants in the preparation will often cause an overestimate of RNA quantity by OD
measurement. It is advisable to submit RNA of the highest quality possible since
degraded/fragmented RNA tends to introduce noise into the sequencing data. Samples not
meeting minimum submission requirements with regard to quantity and quality will result
in a delay to project initiation.
All samples must meet the following criteria:
1. All samples must be treated with RNase-free DNase prior to submission and free of DNA
contamination.
2. RNA must not show signs of degradation as measured by distinct bands of ribosomal peaks
of relative intensities (gel electrophoresis or Bioanalyzer results). Please see example at the
end of the document. The JGI does not require submission of either a gel photo or Bioanalyzer
trace with samples. However, Investigators are strongly advised to assess the quality of
materials to be submitted by gel electrophoresis (including a standard molecular weight marker)
or Bioanalyzer.
3. A260/280 greater than 1.8 (spectrophotometer/NanoDrop). A lower ratio is often indicative of
protein/DNA contamination; a ratio higher than 2.1 may indicate residual guanidine thiocyanate
or beta-mercaptoethanol. Protein contaminants should be re-extracted using
phenol:chloroform:isoamyl alcohol; other contaminants by EtOH ppt. In general, JGI
recommends LiCl extraction for cleaning up RNA.
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4. Quantification must be performed by fluorometric measurement (for example, Qubit
instrument using PicoGreen – Life Technologies, Inc.). Samples that are quantified using a
spectrophotometer or NanoDrop will not be accepted.
The table below should be used as a guide for the amount of total RNA required for the most
common JGI library types:
Library Type
Eukaryotic RNA
Eukaryotic RNA –
low input
Eukaryotic smRNA
(tube)
Eukaryotic smRNA
(plate)
Prokaryotic RNA
Prokaryotic RNA –
low input
Prokaryotic smRNA
Tube
volume
(ul)
Plate
volume
(ul)
Concentration
range (ng/ul)
3000
25-500
25-150
40-1000
300
25-500
-
10-1000
4000
25-500
-
200-1000
4000
-
25-150
166-1000
3000
25-500
-
40-1000
300
25-500
25-150
10-1000
13000
25-500
-
166-1000
Selection Requested
method
mass (ng)
polyA
selection
polyA
selection
rRNA
depletion
rRNA
depletion
The actual amount of total RNA needed may vary by individual project. Please consult with
your Project Manager if you may not be able to provide adequate material.
5. For small RNA, commercially available kits are available for isolation of total RNA containing
miRNA or small RNA (<200bp). Please refer to the manufacturer’s instructions to ensure the
protocol will retain RNA <200 bp.
Shipping:
Prior to shipping samples to the JGI, all sample metatdata must be completed in its entirety. If
you have questions about required information, consult with your Project Manager early in the
submission process.
1. RNA samples are most stable in RNase-free water. RNA should be completely dissolved and
shipped on dry ice. If you have concerns about shipping your material on dry ice, RNAstable
may be an appropriate alternative. Please discuss with your Project Manager.
2. Individual RNA samples should be shipped to the JGI in a single tube (or well of a plate). If
the RNA is prepared in multiple preps, each prep should be QC’ed separately, and good preps
pooled into one tube for shipping to the JGI. You must ship all samples in the barcoded
tubes or plates provided by the JGI.
Last updated 10/17/2016
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3. Do not ship samples until you have received a shipping approval email from the JGI.
Samples that have not been approved will be returned to sender.
Example of Bioanalyzer results of RNA:
Total RNA: Clear 18s and 28s rRNA peaks; no degradation.
Last updated 10/17/2016
2800 Mitchell Drive, Walnut Creek, CA 94598
www.jgi.doe.gov